Obesity is widely recognized as a serious health problem for developed countries, and has reached epidemic status in the United States. According to recent studies, more than 50% of the U.S. population is considered overweight, with more than 25% diagnosed as clinically obese and at considerable risk for heart disease, non-insulin dependent diabetes mellitus (NIDDM), hypertension, and certain cancers. This epidemic presents a significant burden on the health care system as projected obesity treatment costs of more than $70 billion annually are expected in the U.S. alone. Strategies for treating obesity include reduction of food intake and enhancing the expenditure of energy.
Neuropeptide Y (NPY), a 36 amino acid peptide neurotransmitter, is a member of the pancreatic polypeptide class of neurotransmitters/neurohormones which has been shown to be present in both the periphery and central nervous system. NPY is one of the most potent orexogenic agents known and has been shown to play a major role in the regulation of food intake in animals, including humans.
Five neuropeptide Y receptors (NPY), the Y1-, Y2-, Y3-, Y4, and Y5- and Y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors (GPCR). The NPY Y2 receptor (Y2R) is a 381 amino-acid which inhibits the activation of adenyl cyclase via Gi while having low homology with other known NPY receptors. There is a high degree of conservation between rat and human Y2 receptor with 98% amino acid identity.
The Y2R receptor is widely distributed within the central nervous system in both rodents and humans. In the hypothalamus, Y2 mRNA is localized in the arcuate nucleus, preoptic nucleus, and dorsomedial nucleus. In the human brain, Y2R is the predominant Y receptor subtype. Within the arcuate nuclease, over 80% of the NPY neurons co-express Y2R mRNA. Application of a Y2-selective agonist has been shown to reduce the release of NPY from hypothalamic slices in vitro, whereas the Y2 non-peptide antagonist BIIE0246 increases NPY release. These findings support the role of Y2R as a presynaptic autoreceptor that regulates the NPY release and hence may be involved in the regulation of feeding. (Poter, E. K., et al. Eur. J. Pharmac. 267, 253-262 (1994)).
Peptide YY3-36 (PYY3-36) is a 33 amino acid linear peptide having specific neuropeptide Y2 (NPY2R) agonist activity. It has been demonstrated that Intra-arcuate (IC) or Intra-peritoneal (IP) injection of PYY3-36 reduced feeding in rats and, as a chronic treatment, reduced body weight gain. Intra-venous (IV) infusion (0.8 pmol/kg/min) for 90 min of PYY3-36 reduced food intake in obese and normal human subjects 33% over 24 hours. These finding suggest that the PYY system may be a therapeutic target for the treatment of obesity. (Bloom, S. et al, Nature Vol. 418, 8 Aug. 2002, P. 650-654). Further, a Cys2-(D)Cys27-cyclized version of PYY, in which residues 5-24 were replaced by a methylene-chain of 5 to 8 carbons in length, showed activation of the intestinal PYY receptor, as evidenced by reduced current across voltage-clamped mucosal preparations of rat jejunum. (Krstenansky, et al. in Peptides, Proceedings of the Twelfth American Peptide Symposium. J. Smith and J. Rivier Editors, ESCOM. Leiden Page 136-137).
Further, covalent modification of proteins with poly(ethylene glycol) or poly (ethylene oxide) (both referred to as PEG), was demonstrated with superoxide dismutase (Somack, R, et al., (1991) Free Rad Res Commun 12-13:553-562; U.S. Pat. Nos. 5,283,317 and 5,468,478) and for other types of proteins, e.g., cytokines (Saifer, M G P, et al., (1997) Polym Preprints 38:576-577; Sherman, M R, et al., (1997) in J M Harris, et al., (Eds.), Poly(ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680 (pp. 155-169) Washington, D.C.: American Chemical Society).
A need exists, however, for novel engineered analogs of PYY having significantly lower molecular weight, while possessing equal or better potency, pharmacokinetic properties and pharmacological properties as existing Y2R agonists. A need also exists for pegylated analogs of PYY in order to, for example, increase peptide half-life and reduce immunogenicity in subjects in need of such agonists.